In the third grant cycle of leptin transport across the BBB, we will focus on the role of leptin receptor (ObR)-positive astrocytes in relaying leptin from blood to the CNS after crossing the blood-brain barrier (BBB). We hypothesize that astrocytes not only regulate leptin transport as vital components of the BBB, but also modulate neuronal leptin signaling by enabling a more rapid onset and faster termination of leptin action in neurons. The cellular studies with primary astrocytes from mice will determine the effects of reactive astrogliosis on subtypes of leptin receptor (ObR) expression and leptin turnover. The mouse studies will test the role of astrocytic activity and astrocytic ObR on CNS kinetics of leptin distribution, cellular signaling, and development of obesity. In Aim 1, we will test the hypothesis that reactive astrocytes facilitate the turnover of leptin in the brain by accelerating intracellular degradation of leptin. In Aim 2, we will test the hypothesis that reactive astrocytes both in culture and in mice with adult-onset obesity show an imbalance of ObR subtypes resulting from differential regulation. Aim 3 will focus on regulatory changes in adult mice with diet-induced obesity or the Avy mutation, both of which exhibit regional specific increases of astrocytic ObR. By use of glial metabolic inhibitors and newly generated astrocyte-specific ObR knockout mice, we will show that these ObR(+) astrocytes play an essential role in the regulation of neuronal leptin signaling. The results will provide the first evidence of the functions of ObR(+) astrocytes in linking BBB transport to the CNS response to leptin. An understanding of the consequence of astrogliosis and upregulation of astrocytic ObR in obesity should enable the targeting of astrocytes to counteract the neuroendocrine dysregulation in obese subjects. PUBLIC HEALTH RELEVANCE: Leptin is a hormone mainly produced by fat tissue. Hyperleptinemia is seen in the metabolic syndrome. Obesity and its associated hyperlipidemia, cardiovascular complications, cancer, and sleep apnea have a rapidly increasing prevalence in the US and many other parts of the world. This study will mainly focus on how astrocytes participate in delivering leptin from blood to brain and modulating its actions on neurons, the most commonly considered effector cells. Astrocytes are the most abundant cells in the brain, but very few studies have addressed whether they have anything to do with leptin and obesity. We recently found that both the mRNA and protein of leptin receptors are indeed present in astrocytes. Moreover, the expression level of these leptin receptors increases in mouse models of adult-onset obesity. This suggests an important role of the astrocytic leptin system in the regulatory changes in obese subjects. It is possible that it is neuron-glial interactions, rather than direct activation of neurons, that play important mediatory roles for blood-borne leptin. Thus, the relevance lies in (a) better understanding of how astrocytes affect obesity onset and progression; (b) better understanding of cell-cell interactions in the brain; and (c) potential identification of novel therapeutic targets to better combat obesity.